The rate-limiting step in steroidogenesis is the conversion of cholesterol to pregnenolone, cholesterol side-chain cleavage (SCC). This enzyme reation occurs on the matrix side of the inner mitochondrial membrane. Cholesterol must be delivered to and pregnenolone removed from the active site. To study this phenomenon in detail, the guinea pig, a cortisol producer like the human being, has been employed as an animal model. A. Uptake of radioactive cholesterol and cholesterol linoleate. Both compounds were found to be taken up at different rates in the inner and outer zones of the adrenal cortex, with about 2-fold greater uptake in the outer zone. The time-course and subcellular distribution after uptake were also analyzed. B. Ginding of lipoproteins to membrane receptors. The guinea pig utilizes low-density lipoproteins (LDL) for the bulk of cellular cholesterol uptake. Plasma LDL of the guinea pig was isolated by flotation, iodinated by the ioding monochloride method and the 125I-LDL utilized to examine LDL-receptor function, including the effect of ACTH on LDL binding. C. Intracellular metabolism of cholesterol. In addition to the large amount of cholesterol present in cellular membranes, the adrenal cortex stores substantial cholesterol (primarily esterified) in large cytoplasmic lipid droplets. The activities of the enzymes responsible for esterifying and de-esterifying cholesterol, viz, acetyl CoA: cholesterol acyl transferase and cholesterol hydrolase were determined. D. Cytoplasmic SCC stimulator and steroid-binding proteins. Several specific steroid-binding proteins have been detected and analyzed such as cholesterol, pregnenolone (PBP), and pregnenolone sulfate-binding proteins. The PBP is the only one to be purified to date. However, repeated efforts to generate antibodies to the PBP have so far been unsuccessful. The pregnenolone sulfate and cholesterol-binding proteins, as well as, the ACTH-inducible soluble stimulator of cholesterol SCC will be purified.